Malaria in human beings is caused by four species of Plasmodium, namely by P. falciparum, P. vivax, P. ovale and P. malariae. According to a report of the World Health Organisation (WHO) from the year 1986, there are worldwide almost 100 million cases of malaria infection. Of these about 1 million, mostly cases of young children which are infected with P. falciparum, are fatal. Because of the appearance of drug-resistant parasites and of insecticide-resistant mosquito vectors, malaria is spreading (Bruce-Chwatt, Essential Malariology, 2nd edition, Heinemann, London [1986]).
Recent technical advances have raised hopes that it would soon be possible to produce an antimalaria vaccine which would counteract the growing spread of malaria. Firstly, new methods in the development of malaria vaccines, e.g. the cloning of genes and their expression in microbial host organisms as well as the use of monoclonal antibodies for antigen identification, can be used. Secondly, long-term cultures of P. falciparum in human red blood cells (Trager et al., Science 193, 673-675 [1976]) have provided a ready source of material for the study of the malaria parasite. More recently, it has become possible to maintain all stages in the life cycle of the parasite in the laboratory (Ponnudurai et al., Trans. R. Soc. Trop. Med. Hyg. 76, 812-818 [1982]; Mazier et al., Science 22.7, 440-442 [1985]).
The natural life cycle of P. falciparum has three different stages. In the first stage, mosquitoes introduce sporozoites into the blood vessels of vertebrates during the intake of food. These sporozoites travel via the bloodstream to the liver and invade the hepatocytes of the host. In the second stage, merozoites develop from these sporozoites. These merozoites pass through several multiplication cycles in erythrocytes of the host and then develop to gametocytes. The gametocytes, which are the sexual stage of the parasite, are taken up by mosquitoes when they feed. After fertilization in the stomach of the insect the gametocytes develop into sporozoites which then travel to the salivary glands of the insect. From there the cycle can begin again.
Sporozoites, merozoites and gametocytes have different antigens (Scaife, Genetic Engineering 7, 57-90 [1988]). Vaccines can be produced in principle against any of the different stages of the malaria parasite. Various merozoite antigens have been used to induce immunity against malaria. None of these antigens has been found to be the ideal vaccine candidate. Here we present a novel schizont/merozoite antigen of the malaria parasite. This antigen is recognized by two monoclonal antibodies (MABs), MAB 2.13 and MAB 7.12, described by Hall et al. (Mol. Biochem. Parasitol. 7, 247-265 [1983]). MAB 2.13 strongly inhibits invasion of erythrocytes by malaria parasites. Western blot analysis (Towbin et al., Proc. Natl. Acad. Sci. USA 76, 4350-4354 [1979]) of protein extracts from whole parasites using MAB 2.13 revealed 4 major bands with a relative molecular mass of 65 kD, 70 kD, 78 kD and 80 kD, respectively, whereby 1 kD equals 1,000 daltons. Affinity purification of the protein extract from whole parasites followed by gelelectrophoretic analysis revealed two additional bands with a relative molecular mass of 40 kD and 42 kD. Most likely the natural form of the antigen recognized by MAB 2.13, the so-called 2.13 antigen, is the 80 kD species, whereas the other bands represent processed forms of this antigen. Whether the processed forms of the antigen occur also in nature, viz. in the parasite, is not clear. Immunofluorescence studies showed that the antigen recognized by MAB 2.13 is associated with the rhoptry organelles of Plasmodium merozoites. More specifically the MAB 2.13 recognizes a determinant or epitope on a polypeptide associated with the rhoptry organelles of Plasmodium merozoites. The rhoptries of Plasmodium are a pair of pear-shaped, electron-dense bodies situated at the apical end of the merozoite. Electron microscopic studies have implicated these organelles as having a key role in the invasion of erythrocytes by the parasite. It was found that by blocking the said determinant the MAB 2.13 is able to inhibit erythrocyte invasion. The other monoclonal antibody MAB 7.12 has similar properties to MAB 2.13 but does not block erythrocyte invasion as efficiently as MAB 2.13.
Campbell et al., Am. J. Trop. Med. Hyg. 33, 1051-1054 (1984) have described rhoptry-associated antigens of 78, 63, 42 and 40 kD present in protein extracts from the El Salvador and Malaysian strains of the P. falciparum parasite. Howard et al., Am. J. Trop. Med. Hyg. 33, 1055-1059 (1984) found proteins of 82, 70, 67, 39 and 37 kD in an immunoprecipitate from merozoites of a Vietnamese strain of P. falciparum. Schofield et al., Mol. Biochem. Parasitol. 18, 183-195 (1986) described four monoclonal antibodies recognizing antigens present in rhoptries of P. falciparum merozoites. Western blot analysis of a parasite extract using these antibodies yielded protein bands of 80, 66 and 42 kD. Two of the monoclonal antibodies described by Schofield et al. are capable of inhibiting the invasion of erythrocytes by merozoites in vitro. Clark et al. (Parasitol. Res. 73, 425-434 [1987]) have described two proteins having a molecular weight of 82,000 daltons and 65,000 daltons, respectively associated with the rhoptry organelles of Plasmodium falciparum merozoites. Braun-Breton et al. (Nature 332, 457-459 [1988]) have shown that cleavage of a phosphatidylinositol membrane anchor activates a serine protease associated with the soluble form of a Plasmodium falciparum membrane protein of a relative molecular mass of 76,000 dalton present in merozoite or isolated schizont membranes. In vitro experiments have shown that this serine protease appears to be involved in the process of red blood cell invasion by malaria merozoites and plays an important role in merozoite maturation. There is no experimental evidence, however, that the proteins described in the above references are in any way related to the protein recognized by previously described MAB 2.13 or 7.12. In addition, it is not known whether the proteins described in the above references are capable of inducing antibodies in a mammalian host which are capable of inhibiting the invasion of erythrocytes by the merozoite form of the malaria parasite in vivo. In contrast, Applicants have shown for the first time that the 2.13 antigen and, thus, the novel polypeptides of the present invention, are capable of inducing antibodies in a mammalian host which are capable of inhibiting the invasion of erythrocytes by the merozoite form of the malaria parasite in vivo.